The main focus of the laboratory in 2003-4 was in elucidating structural mechanisms of lipid and phophoinositide signaling and protein trafficking. New insights into lipid droplet coating by PAT family proteins The Perilipin/ADRP/TIP47 (PAT) proteins localize to the surface of intracellular neutral lipid droplets. Perilipin is essential for lipid storage and hormone-sensitive regulated lipolysis in adipocytes, and perilipin-null mice exhibit a dramatic reduction in adipocyte lipid stores. A significant fraction of the ~200 amino acid N-terminal region of the PAT proteins consists of 11-mer helical repeats that are also found in apolipoproteins and other lipid-associated proteins. The C-terminal 60% of TIP47, a representative PAT protein, comprises a monomeric and independently folded unit. The crystal structure of the C-terminal portion of TIP47 was determined and refined at 2.8 ? resolution. The structure consists of an alpha/beta domain of novel topology and a four-helix bundle resembling the LDL receptor-binding domain of apolipoprotein E. The structure suggests an analogy between PAT proteins and apolipoproteins in which helical repeats interact with lipid while the ordered C-terminal region is involved in protein:protein interactions. The structure reveals a small hydrophobic pocket on the protein surface where the helical bundle and alpha/beta domains meet. The physiological function of this pocket is probably to bind lipid monomers. The size, shape, and hydrophobicity of the pocket suggest it as a possible target for small molecules which could be potential new anti-obesity therapeutics. Structure of the catalytic core of inositol 1,4,5-trisphosphate 3-kinase Soluble inositol polyphosphates are ubiquitous second messengers in eukaryotes, and their levels are regulated by an array of specialized kinases. The structure of an archetypal member of this class, inositol 1,4,5-trisphosphate 3-kinase (IP3K), has been determined at 2.2 ? resolution in complex with magnesium and adenosine diphosphate. IP3K contains a catalytic domain that is a variant of the protein kinase superfamily, and a novel four-helix substrate-binding domain. The unique helical domain of IP3K blocks access to the active site by membrane-bound phosphoinositides, explaining the structural basis for soluble inositol polyphosphate specificity. Structure of the ESCRT-II endosomal trafficking complex The multivesicular-body (MVB) sorting pathway plays crucial roles in growth-factor-receptor downregulation, developmental signaling, regulation of the immune response and the budding of certain envelope viruses like human immunodeficiency virus (HIV). Ubiquitination is a signal for sorting into the MVB pathway, which also requires the functions of three protein complexes, termed ESCRT-I, -II and -III (endosomal sorting complex required for transport). We determined the crystal structure of the core of the yeast ESCRT-II complex, which contains one molecule of Vps22, the C-terminal domain of Vps36, and two molecules of Vps25, and has the shape of a capital letter Y. The N-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin binding NZF domain of Vps36 both protrude from the tip one branch of the Y. Vps22 and Vps36 form nearly equivalent interactions with the two Vps25 molecules at the center of the Y. The structure shows how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of Ub-cargo from one complex to the next.